IL37312A - The extraction of an anti-hypertensive principle from anacardium occidentale l and pharmaceutical compositions containing it - Google Patents

Description

iniR B* »ao η η η »^*s?5i»i % nVe/:me.pTR Tim extraetioii of an s-a ix-iiyper isaslw rincifi© fro® emeax& m oecideots^ 1 and !iarmsem icaX <»mposi t$as 4 The present invention relates to extracting an anti-hypertensive principle from the bark of Anacardium occidentale L.

Anacardium occidentale L. , the cashew tree, is a tree of North American origin, planted widely in Senegal. The fruit, the anacard, cashew or cashew nut, has a certain number of external medical uses, such as the treatment of ulcers and verrucas. The main therapeutic use of the fruit is in topical anti-leprosy treatment. The juice of the mesocarp is allowed to run onto the leprous spots and anti-leprosy ointments are applied to the artificially produced sores produced by the juice.

It is known from the publication of AGUIAR et al in the Brazilian review "Anals da Paculdade da Medecina da Universidade do Recife", volume 18, Ho. 2, pages 193-7 (1958) " that the bark of Anacardium occidentale L. contains an active hypoglycaemia-inducing agent, which is obtained by treating powdered anacardium bark with boiling water (decoction), the latter activity being demonstrated in rats treated with a decoction of 50 g. of bark per litre of water. No anti-hypertensive action alongside the hypoglycaemia-inducing activity of the extract produced in accordance with such a process has been discovered.

Israel Patent No. 34901 ' In our θβρea4¾Hg-¾?i¼ie¾r-P-a^kest--Ap^iGati-G»--Ho-» 3&,i\ 2?--Q-f— 3r thr-3¾3ry-i96 » we &av© described an extraction process which is different from that of De AGUIAR, according to which the bark of Anacardium occidentale L. is macerated with water, in the absence of light and at a low temperature, preferably between about 0 and °C The extract obtained by dipping the bark in cold water and heating the latter to the boil showed no anti-hypertensive activity whatsoever; this indicates the need to carry out the process at a low temperature and in the dark to obtain an anti-hypertensive extract.

The present invention provides a new process for the extraction of Anacardium occidentale L, which yields a much purer product than that obtained according to the Israel Patent process of our aforesaid Bg¾t¾eh-Αρρίίcation. It has been found, surprisingly, that the active anti-hypertensive principle is contained in the tannins of the bark. The new process therefore relates to the isolation of the purified anti-hypertensive active principle from the tannins of the bark.

The new process for obtaining an extract of bark of Anacardium occidentale L. possessing an anti-hypertensive action comprises: (a) subjecting the ground bark to at least one extraction with petroleum ether; (b) after removing the petroleum ether, subjecting the insoluble residue which is left to at least one extraction with diethyl ether; (c) after removing the diethyl ether, subjecting the insoluble residue which remains to at least one extraction with ethanol, methanol, a mixture thereof, or a mixture of either or both with water, (d) after removing the insoluble residue and concentrating the soluble fraction according to (c) in vacuo to produce a dry extract, dissolving the said dry extract in water, and collecting the anti-hypertensive principle by precipitation with an alkaloid or hide powder, each of the extractions according to (a), (b) and (c) being carried out in the absence of light and at a low temperature, and the process of concentration in vacuo being carried out at a temperature below 6Q°C.

In the process of the invention, the ground bark of Anacardium occidentale L. is treated with solvents of increasing polarity. In the first place, the waxes and greases contained in the bark are removed by treatment with petroleum ether followed by diethyl ether. The insoluble residue which remains is extracted with a solvent which makes it possible to dissolve the tannins contained in the starting material. Various solvents, such as water, alcohols and acetone are known for the purpose of dissolving the tannins. In the present case, it has been found that acetone does not permit the active principle to be extracted in good yield.

The minimum duration of each extraction is 3 hours. It is desirable, especially in stage (a), for the duration of treatment to exceed the minimum duration if the bark is not finely ground. Under such circumstances it is on the whole preferable that the duration of extraction with petroleum ether should be at least 8 hours in order to obtain good yields.

According to a preferred embodiment of the new process: (a) 20 to 80 g. of bark are treated with 1 litre of petroleum ether, (b) the fraction which is insoluble in the petroleum ether is treated with 1 litre of diethyl ether, and (c) the fraction which is insoluble in the diethyl ether is treated with 1 litre of ethanol, methanol, a mixture thereof or a mixture of either or both with water, and (d) after removing the insoluble residue which remains and concentrating the soluble fraction according to (c) in vacuo at a temperature below 60°G., to obtain a dry extract, the said extract is dissolved in water and the active principle is collected by precipitation with caffein or hide powder, each of the extractions (a), (b) and (c) being carried out in the absence of light, at a temperature of between 0 and 15°C«» preferably between 2 and and for at least 3 hours.

The solvent preferred at stage (c) for dissolving the tannins is a water-ethanol or water-methanol mixture containing 35 to 85%s and preferably 7 %» by volume of the alcohol.

To precipitate the tannins containing the antihypertensive fraction, either an alkaloid or hide powder is used. Amongst the alkaloids, good results have been obtained by reacting caffein with the dry extract obtained by concentrating the soluble fraction according to (c).

Preferably 5 parts by weight of the said dry extract are dissolved in water and reacted with an aqueous solution of caffein containing about 1.32 parts by weight of caffein. In general, 5 g» of the said dry extract are dissolved in solution containing 12 g. of caffein/1.

The precipitate thus obtained is redissolved and the caffein is extracted with chloroform* Preferably, the said precipitate is dissolved in the minimum amount of methanol (about .10 to 20 ml. per 5 g. of the said dry extract), water (250 ml. for the amounts indicated above) is added, and three extractions are carried out with chloroform (using, in each case, 10 to 100 ml, preferably 15 to 0 ml., of chloroform for the amounts indicated above).

The methanol-water phase is then concentrated in vacuo at a temperature below 60°C, until dry. To purify the powder thus obtained, it is redissolved in the minimum amount of methanol and precipitation is effected by adding ether (CH^OCH^). This purification is aimed at reducing the toxicity of the active anti-hypertensive principle, as will be shown later.

Hide powder is a reagent used by tanners to characterise tannins (which are adsorbed by hide powder). To recover the tannins which have been adsorbed, a treatment on an ion exchange resin is preferably carried out, the product thereafter being purified, if necessary, as described for the extract of the methanol-water phase.

The successive operations mentioned above are the result of simultaneous chemical and pharmacodynamic research which have made it possible, first to follow the active principle in the various extracts, and secondly to detect any possible degradation, dilution or concentration of the active principle during the treatments to which it is subjected, as compared with the aqueous extract, containing 50 g. per litre, obtained by maceration in Israel Patent accordance with the Brar½-&k-Appliea*iea mentioned above.

The barks were successively exhaustively extracted with solvents of increasing polarity and anti-hypertensive properties of the various extracts were assessed by the following two methods: 1) Indirect method By oral administration to, or by injection into, normal rats, carotid-hypertensive rats, and renal-hypertensive rats; 2) Surgical method Under an anaesthetic, using a carotid cannula, and without anaesthetic, using a cannula left in the abdominal aorta, all the rats used weighing between 250 and $00 g.

The present invention also comprises pharmaceutical compositions useful in anti-hypertensive therapy, comprising an anti-hypertensive fraction obtained from Anadardium occidentale L. by the procedure described above, in association with an inert pharmaceutically acceptable carrier.

The following Examples illustrate the invention.

EXAMPLE 1 12 g. of bark of Anacardium occidentale L. , ground in a mixer, or defibred and powdered, are treated with 200 cur of petroleum ether for 8 hours at 20,, to "degrease" it. A filtrate (A) and an insoluble residue (B) are obtained. 0.053 g. of a pasty powder is recovered from the filtrate (A) by concentration in v¾cuo at a temperature below 60°C. This powder is soluble in petroleum ether.

The residue (B) which is insoluble in petroleum ether is extracted with 200 cm of diethyl ether for 8 hours at 2°C, in the dark. A filtrate (C) and an insoluble residue (D) are obtained. 0.084 g, of product are recovered from the filtrate (C) by distilling the diethyl ether at a temperature below 60°C. in vacuo.

The residue (D) which is insoluble in diethyl ether is extracted with a 75% by volume aqueous solution of ethanol for 24- hours at 2°C. in the dark. A filtrate (E) and an insoluble residue (P) are obtained. After evaporation of the filtrate (E) in the dark, in vacuo, at a temperature below 60°C, 4·.32 g. of brown powder (ET) are obtained.

On extraction of the residue (1?) , which is insoluble in 75% strength ethanol solution, with 200 cm^ of water for 24- hours in the dark at a temperature of 2°C, it is possible to recover 1.22 g. of a dark brown powder after distilling the solvent under reduced pressure at a temperature below 60°C.

The extracts obtained after the extractions with petroleum ether, diethyl ether and 75% strength ethanol were tested in rats as indicated above and compared with the aqueous extract, containing 50 g. per litre, obtained by maceration for 24- hours in water in accordance with Israel No. 34901 the Patent Αρρϊ±ββ*±θη-ϊίθ-·—§&j 2-?, referred to above· The powders obtained from the filtrates (A) and (B) and from the residue (F) proved inactive when administered to rats. Conversely, extract ET, in an aqueous solution containing 20 g/1, gave positive results on injection of 0.1 to 0.3 CBT of the said aqueous solution.

The aqueous solution, containing 50 g/1, of the extract obtained by aqueous maceration in accordance with Israel Patent the aforesaid S½*i«¾--S>e½ii-ea¾-9fi also gives good results; in rats, a drop of 1 to 2 cm Hg. is observed for about twenty minutes after administration of a dose of 1 ml/kg of the said solution.

On replacing the 75% ethanol with 75% t>7 volume methanol, it was found that the activity in rats of the soluble extract in the said solution is substantially the same. In general, one-third of the weight of bark of Anacardium occidentale L. is obtained from the extract with 75% ethanol.

Furthermore, it has been observed that starting from some of the dry extracts obtained by the process described above, certain compounds precipitated spontaneously in alcoholic solution as soon as the said dry extracts are dissolved. Thus a quantity of the soluble extract obtained in stage (c) with ethanol was dissolved in methanol. After the solution has stood and the precipitate then recovered by centrifuging, the product obtained was tested in rats. It was sparingly soluble in water, and proved inactive at a concentration of 3 g/l« t Equally, starting from extract ET, crystals were sometimes recovered in an identical manner which, had a salty taste, were sparingly soluble in water, and were inactive in rats at a concentration of 3 g/1 in water, Extract ET, freed from these impurities, is stable. When stored in the dark, it shows substantially the same activity after several months.

A quantity of the extract ET was dissolved in methanol and the solution left to stand for at least 24- hours. The precipitate obtained was recovered by centrifuging. Crystals having a salty taste were also recovered from the extract obtained with 75% ethanol.

These two products, which are sparingly soluble in water, were tested on rats at a concentration of 3%°j an proved inactive .

Study of the activity in rats made it possible to establish that generally the extract obtained with 75% ethanol, at a concentration of 2% based on the solids content of the extract, gives positive results on injection of 0.1 to 0.3 cur of solution.

The aqueous extract of 5 strength based on the weight of dry bark also gives good results: a decline of 1 to 2 cm Hg. occurring over about twenty minutes.

Summarising, the extract obtained with 75% strength ethanol (ET) is active at a concentration of 2% when 0.1 to 0.3 cur is injected into rats weighing 250 to 300 g.

The active principle appears to be relatively stable; when stored in the dark, it shows substantially the same activity after 1 to 2 months.

The extraction according to Example represented schematically in Table I below.

TABLE I Bark of Anacardium occidentale L : 12 g 200 cur of petroleum ether Anacardium 0.053 g» of petroleum ether extract of diethyl ether extract inactive Anac *ar—dium 4.32 g. of 75% strength) active ethanol extract 200 cnr of water v£ Anacardium 1.22 g. of water extract : inactive 2) Purification stage - Purification of the ethanol fraction ET In view of the preceding results, attempts were made to separate a tannins fraction from a non-tannins fraction. For this purpose, tannins were precipitated with a solution of caffein.

The tannins in 5 g« of ET, dissolved in 30 ml. of water, are precipitated by adding 110 ml. of a solution of 12 g* of caffein/1. The precipitate obtained is centrifuged or filtered off, washed with water and dissolved in the mini-mum amount of methanol (about 12 ml). This solution of tannins in methanol is made up to 250 cnr with water and extracted three times with chloroform to free it of caffein. The aqueous methanolic solution obtained is evaporated in vacuo. 2.80 g. of powder (T.A.N.) are obtained. The chloroform solutions are evaporated. 1.06 g. of caffein are recovered for the tannins and 0.120 g. of caffein for the non-tannins, representing a recovery of 1.18 g. of caffein for 1.5 g. employed.

The filtrate is an aqueous solution which in principle is free of tannins. This solution is extracted with chloroform and evaporated in vacuo. 1.60 g. of powder (S.T.A.N.) are obtained.

Balance: With 5 g. of Anacardium occidentale L bark, 2.8 g. of "tannins" and 1.60 g. of "non-tannins" are obtained, in accordance with a fractionation which can schematically be represented in Table II below.

TABLE II Residue Filtrate Extraction of caffein Centrifuge - wash with chloroform (0.120 g Dissolve in a minimum of of caffein) methanol Concentrate in vacuo. Dilute with water: 250 ml Dry extract: 1.60 g 3 extractions with chloro< form to remove the tiON-ANTI-HYPERTENSIVE FRACTION caffein (1.06 g) Aqueous-m Φethanolic solution Evaporate in vacuo Dry extract: 2.80 g ANTI-HYPERTENSIVE FRACTION This method of fractionation makes it possible to recover a "tannins" fraction and a "non-tannins" fraction without adulterating the solutions with other products, because the caffein is removed.

Physiological experiments relating to the activity of the various extracts were carried out.

An aqueous solution of "non-tannins" S..T.A.N, at a concentration of 30 g/1 was injected intravenously into rats at doses of 0.1 to 0.3 cm , and does not cause any lowering of (blood) pressure.

The aqueous solution containing 10 g. of tannins T.A.N./I is very active when injected into rats at doses of 0.1 to 0.2 cur5.

The solution containing 10 g. of E.T./l under the same conditions has practically no effect. An aqueous solution of 20 g. of extract ET/1 shows a good activity when injected into rats at doses of 0.1 to 0.2 cm , It follows from the series of experiments with the solutions of extracts S.T.A.N., T.A.N, and ET that the active principle behaves like a tannin. 3) Purification of the tannin extract T.A.N.

The solid extract T.A.N, is redissolved in the minimum amount of methanol (about 10-12 ml) and then reprecipitated with dimethyl ether. The ether is added dropwise to the methanol solution until the product has precipitated completely. The mixture is filtered. Dry weight: 2.50 g.

If the T.A.N, extract is compared with that obtained by dissolving in methanol and precipitating with ether, the same activity is observable, but with a reduction in toxicity when injected intreperitoneally into the animal. This may be due to the fact that an inactive product dissolves in the ether. In particular, the absence of oxidised forms is observed.

The difference in toxicity is illustrated in Table III below: TABLE III Nature of the extract injected LD-50 in mice, when as a 10 g/1 aqueous solution administered intraperitoneally Dry extract according to Israel Patent No. 34901 Dri¾iah-A^^ire ^en-Me· 135 mgAg Extract ET 280 mgAg Extract T.A.N, after purification in accordance 600 mgAg with Example 1, Section 3 Table III shows first that on purification by precipitation with caffein followed by the treatment according to Example 1, Section 3, the ID- 50 changes from 280 mgAg to 600 mgAg and, secondly, that the process of the invention represents a technical advance because the final product obtained is much less toxic than the extract obtained by aqueous maceration in Israel Patent accordance with the Ba?arl5ieh--Sp The same process was carried out on a solution of 0.550 g. of "tannins" (T.A.N.) in 100 cm5. After filtration and evaporation, 0.024 g. of sparingly water-soluble "non-tannins" were found, and this material did not show an activity in aqueous solution at a concentration of g/1.

In order to obtain the tannins adsorbed on the hide powder, an ion exchange resin can be used; the tannins fraction thus recovered is dried in vacuo at a temperature below 60°C., and can then be purified by the process of Example 1, Section 3· The various experiments described above show that the active fraction behaves like a tannin because it is adsorbed by the hide powder and is precipitated by the alkaloids.

In order to study the anti-hypertensive action, experiments were carried out on normal rats and on rats rendered hypertensive by three different types of experimental hypertension (renal, metacorticoid and neurogenic). The extract obtained according to Example 1, Section 3> was administered orally, at a dose of 40 mgAg, to batches of 8 to 1 animals.

The arterial pressure is recorded before forced feeding and thereafter every hour. It drops slowly, with a maximum action at between 2 and 5 hours after administration, followed by a rise between 5 a 9 hours, in accordance with the expected progression.

The normal control animals show little reaction.

The neurogenic hypertensive rats showed the greatest drop in pressure, the average blood pressure falling 4.2 cm i 0.5 cm.Hg. over the course of about 9 hours.

In the metacorticoid hypertensive rats, an average reduction in blood pressure of 3·1 - 1·3 cm.Hg. was observed.

In the renal-hypertensive rats a reduction in average blood pressure of 3·6 i 0.5 cm.Hg. was observed.

In general, the rats withstood the administration of the product well. Some of the results obtained are shown graphically in accompanying Figures 1 and 2.

Figure 1 represents the average curve of the arterial blood pressures of 8 renal-hypertensive rats which were orally (forced feeding) given 0.4 ml. (40 mg/kg) of an aqueous solution containing 20 g/1 of the extract obtained according to Example 1.

Figure 2 shows the average curve of the arterial blood pressures of 13 renal-hypertensive rats after intraperitoneal injection of 0.2 ml. (20 mg/kg) of an aqueous solution containing 20 g/1 of the extract obtained according to Example 1, Section 3· In Figures 1 and 2, the arterial blood pressure is expressed in cm.Hg. (as ordinates) as a function of the time in hours (as abscissae).

Furthermore, the same tests of anti-hypertensive activity were carried out on renal-hypertensive monkeys. As was to be expected, the anti-hypertensive action is much greater in experimentally hypertensive monkeys than in normal monkeys.

The extract according to the invention, as a g/1. aqueous solution, was administered orally at a dose of 3 mg/kg. to 8 hypertensive monkeys for a period of 7 days. The arterial blood pressure was measured before the start of the treatment and then daily at 3 and 5 hours after administration (effected by forced feeding) for the entire duration of the treatment. At the end of the treatment, lasting 7 lays, the average drop in arterial blood pressure was 3.4- cm. of mercury and the blood pressure started to rise again on the 11th day following the start of the treatment.

Another series of experiments carried out on renal-hypertensive monkeys are summarised in Figure 3· These animals received, by forced feeding, 10 ml. of an aqueous solution of 20 g/1. of the extract according to the invention (dose 0 mg/kg.). The duration of the action in hours aftor administration has been shown as the abscissae and the average blood pressure, expressed in cm. of mercury, as the ordinates.

Investigations were also carried out on the mechanism of action of the extract obtained by the process of the invention. The results observed are summarised below.

The cardiac frequency varies little under the influence of the extract. The extract produces little change in adrenaline hypertension. Atropine does not hinder the hypotensive action of the extract and the action appeared not to be due to a muscarinic effect. The hypertensive action persists after lowering of the pressure by injection of 50 mg. of Penthonium.

The anti-hypertensive extracts obtained by the process of the invention may be used in therapy as a composition containing 1 to 95% by weight of active ingredient.

In order to prepare therapeutic compositions from the anti-hypertensive extracts, physiologically tolerated vehicles for solid forms or liquid forms may be used.

Suitable therapeutic preparations in a solid form include powders, tablets, dispersible granules, tablets, capsules, cachets and suppositories. A solid vehicle can consist of one or more substances acting as a diluent, sweetener, solubilising agent, lubricant, binder, suspending agent or disintegrating agent. The vehicle can also be an encapsulating substance.

In the case of powders, the vehicle is a finely-divided solid xtfhich is intimately mixed with the active compound, which is itself finely divided. .Amongst the solid vehicles there may be mentioned magnesium carbonate, magnesium stcarato, talc, sugars such as lactose, pectin, dextrin, starch, gelatine, gum tragacanth, methylcellulose, sodium carboxymethylcellulose, waxes of low melting point, and cacoa butter. By "preparation" there is also understood the formulation of the active product with an encapsulating substance and with a vehicle so as to give a capsule in which the compound (with or without excipient) is coated by the said encapsulating agent.

Preparations in liquid form comprise solutions, suspensions and emulsions. For example, physiological solutions and mixtures of propylene glycol and water may be mentioned for parenteral injection.

For oral use, the active ingredient can be dispersed in water with the incorporation, if necessary, of surface-active agents, such as, for example, gum arabic or methylcellulose. when administered orally, the therapeutic composition prefera.blj' contains 100 mg. to 1 g. of antihypertensive extract combined with a physiologically tolerated vehicle, xhilst when administered by injection the therapeutic composition contains $0 to 300 mg. of active ingredient.

The process of treatment of hypertension in man and warm-blooded animals consists of administering 1 to 5 doses daily of a composition containing 100 mg. to 1 g. of active ingredient if administered orally or 3 to 300 g. of active ingredient if administered by injection, the preferred administration being oral administration in the form of cachets, tablets or gelatine-coated pills, The methods of characterisation of the various compounds contained in the active principle are given below. a) Extract E.T. - Test for tannins 1) With 1% aqueous FeCl^ solution, a white precipitate is obtained, 2) with STIASNY reagent, a curdy pink precipitate is obtained after filtering and adding an excess of sodium acetate, and a slight blue-violet colouration is obtained with FeCl^, 3) with saline gelatine, a white precipitate is obtained.

Those various reactions suggest the presence of a preponderance of condensed tannins. b) Tost or flavonoids The SHIBATA reaction gives an orange colouration in isoamyl alcohol, which suggests the presence of free flavonoids. The same reaction without magnesium filings does not give a cherry-red or violet colouration but an orange colouration probably corresponding to a catechol. c) Test for quinones The maceration of 2 g. of bark with chloroform for 1 hour followed by exhaustive extraction with sodium hydroxide solution does not give a colour. The same reaction carried out after hydrolysis gives an orange- yellow colouration. Those results suggest a substance which after hydrolysis gives the colour reaction of quinones with alkalis. d) The tost for alkaloids and saponins proved negative.

Additionally, tests were carried out to attempt to separate the various extracts "by chromatography on a plate covered with silica gel. Good separation is achieved for E.T. and S.T.A.N, with anisaldehyde as the developer and butanol, acetic acid and water, in the ratio of 60/40/40 by volume as the eluting agent. '3 Well-defined black spots are thus observed, identical for E.T. and S.T. .N., namely a major spot (Rf = 0.54·), an average spot (Rf 0.44) and a slight spot (Rf = 0.04).

The characterisation of the T.A.N, extract purified according to Example 1, Section 3» was carried out by thin layer chromatography (support : silica gel or polyamide gel; eluting agent : butanol-acetic acid). In an alkaline medium (5N KOH) no migration occurs. In an acid medium migration does occur (Rf = 0,65, developer : diazotised p_-nitroaniline) » -furthermore, using a bark powder of which the composition is generally the following: Water 10 to 15% by weight Tannin fraction T.A.N. 19 to 23% by weight Non-tannin fraction (S.T.A.N. ) removed according to the process of the invention 3 to 5% by weight Gums (containing alkaloids), rosin, varnis and wax 57 "bo 68% by weight the presence of the following was observed: a large amount of thiols; reducing sugars or phosphate esters of sugsrs, desoxyribosides and indole rings.

Claims (12)

HE exAXHt

1. Process for obtailning an extract of "bark of -toacardiuc. occidentale L. possessing an anti-hypertensive action, which comprises: (a) subjecting the ground "bark to at least one extraction with petroleum ether, (b) after removing the petroleum ether, subjecting the insoluble residue which is left to at least one extraction with diethyl ether, (c) afte removing the diethyl ether, subjecting the insoluble residue which remains to at least one extraction with ethanol, methanol, a mixture thereof, or a mixture of either or both with water, (d) after removing the insoluble residue and by concentrating the soluble fraction according to (c) in vacuo to produce a dry extract, dissolving the said dry extract in water, and collecting the anti-hypertensive principle by precipitation with an alkaloid or hide powder, each of the extractions according to (a) , (b) and (c) being carried out in the absence of light and at a low temperature, and the process of concentration in vacuo being carried out at a temperature below 60°C»

2. Process according to claim 1 in which (a) 20 to 80 g of bark are treated with 1 litre of petroleum ether, (b) the fraction which is insoluble in the petroleum ether is treated with 1 litre of diethyl ether, (c) the fraction which is insoluble in the diethyl ether, is treated with 1 litre of ethanol, methanol, a mixture thereof or a mixture of either or both with water, and (d) after removing the insoluble residue which remains, concentrating the soluble fraction according to (c) in vacuo at a temperature below 60°0 to obtain a dry extract, the said extract is dissolved in water and the active principle is collected by precipitation with caffein or hide powder, each of the extractions (a) , (b) and (c) being carried out in the absence of light, at a temperature of between 0 and 15°0, preferably between 2 and 5°C, and for at least 3_hours.

3. Process according to claim 2, in which the solvent used in stage (c) is a water-ethanol mixture containing 35 to 85% ethanol by volume.

4. Process according to claim 3 in which the said niixture contains 75% ethanol by volume.

5. Process according to claim 2, in which parts by weight of the dry extract obtained by concentration in vacuo of the soluble fraction according to (c) are reacted in water with about 1.32 parts by weight of caffein,

6. Process according to claim 5} in which g of the said dry extract are dissolved in 30 ml of water and reacted with 110 ml of an aqueous solution of 12 g of caffein1, the precipitate thus obtained is isolated and in that, after washing withwater, the said precipitate is redissolved in the minimum amount of methanol, the solution is made up with 250 ml of water, the caffein is extracted .three times with 10 to 100 ml of chloroform^ and the water-methanol phase is concentrated to dryness in vacuo at a temperature below 60°C.

7. Process according to claim 6, in whicjh the dry extract obtained by concentration of the water-methanol phase is redissolved in the minimum amount of methanol and precipitated by dimethyl ether and in that the said precipitate is collected after removal of the filtrate.

8. Process according to claim 1 substantially as described in the foregoing Examples.

9. The anti-hypertensive extract obtained by the process of any one of claims 1 to 8

10. Therapeutic composition containing 1 to 95% by weight of an anti-hypertensive extract according to claim 9 and a pharmaceutically acceptable vehicle.

11. Therapeutic composition according to claim 10 for oral administration containing 100 mg to 1 g of active ingredient per unit dosage.

12. Therapeutic composition according to claim 10 for administration by injection, containing 30 to 300 mg of active ingredient per unit dosage.